Camel Milk-Clotting Using Plant Extracts as a Substitute to Commercial Rennet

(e transformation of camel milk into cheese is an operation considered very delicate because of several difficulties encountered in achieving coagulation. (e present study aims to improve coagulation abilities of camel milk using enzyme extracts from pineapple, kiwi, and ginger. Our results concerning the characterization of the enzymatic extract showed an extraction yield that varies according to the type of extract (pineapple: 75.28%± 4.59, kiwi: 63.97%± 5.22, and ginger: 28.64%± 1.47). (e optimum coagulation conditions of the 3 types of extract were as follows: for pineapple: pH� 5 and temperature� 45°C; for kiwifruit: pH� 6.6 and temperature� 40°C; and for ginger: pH� 6.6 and temperature� 45°C. A fresh cheese was made from camel milk with a particular nutritional quality and consistency. (e kiwi proteases displayed chymosin-like properties and thus hold the best potential for use as a milk coagulant in cheese production.


Introduction
Camels (Camelus dromedarius) are of particular interest in arid and desert regions. eir unique adaptability makes this species ideal for exploitation facing the challenges of global warming and perfect allies for food security under changing climate. Camel milk is mainly consumed raw just after milking or fermented but rarely processed into cheese [1] due to a low amount of κ-casein [2]. Hence, several studies focused on the functional and coagulation properties of the camel milk's proteins [3][4][5][6]. In spite of the above difficulties, satisfactory cheese can be made when cheese-making procedures are adapted to camel milk's particular characteristics [7]. A lot of work was dedicated to improve coagulum formation process using various proteolytic enzymes obtained from animal, plant, and microbial sources.
In general, bovine chymosin is the most used enzyme in a cheese-making procedure. However, the availability of calf rennet has become limited due to the worldwide increase in cheese manufacture, coupled with the reduction in the slaughter of calves due to their low meat production, and has led to search for alternative milk-clotting enzymes, as appropriate rennet substitutes [8][9][10]. Proteases from plant sources offer a high potential as nonanimal rennet alternative in production of cheese, food (e.g., production of novel dairy products, meat tenderizers, and protein hydrolyzate production), and medicine (e.g., digestive and anti-inflammatory agents) [11,12]. An increasing interest toward milk-clotting agents obtained from plants in cheese industry has been noticed because of their easy availability and simple purification processes [9,13]. Furthermore, the use of plant proteases in cheese manufacturing promotes the greater acceptability by the vegetarians, some religious groups, and exigency of banning genetically engineered food [14]. For years, plant extracts have been used as milk coagulants in traditional cheeses which are mainly produced in Mediterranean countries, West Africa, and Asia [15]. Indeed, dried cardoon flowers of Cynara cardunculus L. and Cynara humilis L. have been used for centuries in the Iberian Peninsula to prepare certain varieties of cheese with a creamy soft-texture and exquisite flavor. e aspartic proteases cardosins and cyprosins (also called cynarases) are the main milk-clotting proteases present in the cardoon extracts [16]. In addition, the juice from the leaves of sodom apple (Calotropis procera) has been used in the African regions of Nigeria and Benin for traditional cheese-making [17], and in the south of China, the rhizome ginger juice is used to produce a ginger milk curd which is highly appreciated due to its tofu pudding-like, sweet-snack characteristics [18]. e common plant proteases papain and ficin (from papaya and fig, respectively) have a low milk-clotting activity/proteolytic activity (MCA/ PA) ratio and have often been mentioned as the principal obstacle to their utilization in cheese-making [19]. Besides texture, the flavor of cheeses may also be affected by the type of plant extract used which leads to bitterness in the final product [14]. Use of fruit extracts such as kiwi (Actinidia L.), pineapple (Ananas comosus L. Mer), and ginger rhizome (Zingiber officinale Roscoe) extract resulted in less off-flavor notes, which are attributed to the production of short peptides that are responsible for bitterness in dairy products when ficin (fig or papaya protease) is used [18]. Moreover, high proteolytic activity has been recorded within the cysteine proteases actinidin (or kiwellin), bromelain (pineapple enzyme), and zingibain (ginger protease) that have potential applications in food industry [18,20].
Hence, the objective of the present study was to evaluate the camel milk-clotting activities of ginger, pineapple, and kiwi extracts in terms of temperature dependence and their performance on yield and textural properties of curds during cheese-making, compared to commercial rennet. ml/l, Chr. Hansen A/S, Hørsholm, Denmark. Fresh camel milk was collected from female camels (Camelus dromedarius) belonging to the Arid Land Institute (IRA Medenine, Tunisia). Samples were brought to the laboratory in an isotherm container and were analyzed and processed upon arrival.

Preparation of Extracts.
Extracts were prepared as described by Mazorra-Manzano et al. [21]. In brief, kiwi, pineapple, and ginger rhizomes were peeled and sliced. en, water extracts were prepared by adding one equal part (w/v) of 20 mM sodium phosphate buffer (pH 7.2). Water extracts were homogenized and then centrifuged at 5000 g for 30 min at 4°C ( ermo Electron LED GmbH Am Kalkberg, Germany) and filtered through a double cheesecloth to remove suspended particles. Fresh extracts were stored at 4°C and were either used the same day for protein and milk-clotting activity determination or frozen at −20°C for further use.
pH and dry matter of enzymatic extract were determined using International standard methods [22]. e protein content was determined according to the Bradford method [23], using bovine serum albumin (BSA) as standard.

Optimization of pH, Temperature, and CaCl 2
Concentrations. Optimum pH and temperature were determined according to the method of Kunitz [24]. To determine the optimal pH for the enzyme activity, 1 ml of plant extract was added to 10 ml of camel milk at different pH values (5, 5.6, 6.2, 6.6, 7.5, and 8.5) at 30°C, and flocculation time (Tc) was measured. In order to determine the optimum temperature, the purified enzyme solution was incubated with casein solution at various temperatures ranging from 30 to 60°C for 10 min in a controlled temperature water bath and the flocculation time was noted. e optimal CaCl 2 concentration was prepared by dissolving 12 g of skimmed milk powder in 100 ml of CaCl 2 solution with a calcium ion concentration range of 0.01 to 0.09 M and added in tubes containing 10 ml of milk at pH � 6.6 and at T � 30°C, and the flocculation time was then determined.

Milk-Clotting Activity.
e milk-clotting activity (MCA) of each extract was determined according to the Berridge method [25] modified by Collin et al. [26] with some modifications. One unit of enzymatic activity or rennet unit (RU) corresponds to the number of units of weight or volume of milk that can be coagulated with 1 ml of coagulant preparation in 100 seconds and at optimal pH and temperature for each plant extract or commercial rennet. e time needed for curd formation was recorded (Tc). MCA was expressed as RU units and calculated as follows: where RU: unit of coagulating activity or rennet unit, V: volume of substrate (ml), Q: volume of coagulant extract (ml), and Tc: flocculation time (sec).

Proteolytic
Activity. Proteolytic activity was determined by the method of Bergere and Lenoir [27] using BSA as a substrate. In brief, 1 ml of 1% protein substrate solution (0.1 M phosphate buffer, pH 7.0) was mixed with 1 ml of coagulant and incubated for 60 min at 35°C. After incubation, the reaction was stopped by the addition of 1 ml of 12% (w/v) trichloroacetic acid. e mixture was vortexed vigorously. e absorbance of the clear filtrate was measured at 280 nm. One unit of the enzymatic activity (U) was defined as the amount of protein that gave an increase of one unit in the absorbance at 280 nm under the described conditions.
2.6. Cheese-Making Process. Fresh camel milk was pasteurized at 65°C for 30 minutes and then cooled to 40°C. e starter culture (Lactococcus lactis) isolated from artisanal fermented milk was then added to decrease pH at 5.5. After about one hour, the enzymatic preparation was then added at the rate of 10% of milk and mixed thoroughly. e mixture was incubated for 24 hours at 37°C. After coagulation, the whey was drained to obtain a fresh cheese and kept at 4°C for further analysis.

Cheeses Characterization.
e physicochemical characteristics were determined using International standard methods [22]. e protein content was determined according to the Bradford method [23], using BSA as standard.
e moisture content (Hm) was calculated according to the following formula: where Hm: humidity (%) and DMC: dry matter content. As microbiological analysis, the total viable counts were determined on a plate count agar (Oxoid Ltd., Basingstoke, UK) at 30°C for 72 h; total coliforms on violet red bile agar (Oxoid) at 30°C for 24 h; mesophilic and thermophilic lactobacilli on MRS agar (Oxoid) at 30°C and 45°C for 48 h under anaerobiosis, respectively; lactococci on M17 agar (Oxoid) at 30°C for 48 h; and yeasts and molds on Worth agar (Oxoid) at 30°C for 72 h. Results were expressed as log colony-forming units per ml of milk or gram of cheese. e fat-soluble and water-soluble vitamins were determined by LC-MS chromatography according to Albala-Hurtado et al. [28].
Minerals in milk and cheese were quantified using Flame Atomic Absorption Spectrometer (ICE 3000 SERIES AA Spectrometer).

Texture Profile Analysis.
Texture properties (TPS) of the cheese samples were determined by a Texture Analyzer Brookfield (model CT-3, MA, USA). Cheeses were carefully cut into pieces (15 × 15 mm) with a cheese slicer and compressed to eliminate the air for 1 hour, and the values have been read directly. 2 parameters were set (penetration speed: 2 m/s; the distance of penetration: 10 mm).

2.9.
Sensory Analysis. Samples of cheese were subjected to sensory evaluation by 42 untrained panellists. Cheese samples were assessed for their taste, color, flavor, acidity, and texture. Panellists received a set of four samples per session, representing cheeses made with different plant extracts and the control made with chymosin. Each sample was evaluated in duplicate. e panellists were asked to drink plain water at the beginning of the sensory evaluation and between samples to try to make the palate conditions similar for each sample. Cheese traits are rated on the basis of 10 cm unstructured lines with 10 points scale (0 � lower intensity and 10 for higher intensity). Scores were the distances (cm) from the left anchor point. At the end of sensory evaluation, panellists were asked to rank overall cheese acceptability.

Statistical Analysis.
e experiments were conducted in triplicate, and data were presented as the mean ± standard deviation. Data related to physicochemical characteristics, TPS, and sensory analysis were subjected to analysis of variance (ANOVA) using the SPSS 20. Duncan's multiple range tests were used to test differences between means with type of rennet as the main factor.

Characterization of Enzymatic Extract.
e physicochemical characteristics of the enzymatic extracts obtained are shown in Table 1. A significant difference was observed in the pH value, dry matter, and proteins between the three types of extract. e highest pH was observed in ginger with a value of 5.24 ± 0.06, pineapple 3.58 ± 0.01, and kiwi 3.46 ± 0.01. e dry matter content was higher for ginger extract (116.15 ± 3.25 g/L) than kiwi and pineapple (88.45 ± 1.55 g/L and 85.15 ± 4.95 g/L, respectively). e kiwi and pineapple had significantly higher protein content (80.58 ± 7.42 g/L and 74.08 ± 1.25 g/L, respectively).

Optimum Conditions of the Enzymatic Extract Clotting
Activity. As shown in Figure 1, the temperature for optimal clotting activity of different extracts studied in this assay was at 45°C for pineapple and ginger and at 40°C for kiwi. e effect of temperature proceeds mainly on the secondary phase of coagulation, which corresponds to the aggregation step. is is due to the importance of hydrophobic interactions in the aggregation of hydrolyzed micelles [29]. e optimal clotting activity was at pH 5 for pineapple extract and 6.6 for kiwi and ginger extracts. Ramet [30] reported that all enzymes used in cheese manufacture are acidic proteases, and their activity is generally optimal at pH values close to 5.5. Few studies on milk-clotting substitutes for calf rennet applied to camel milk are available. Our findings differed from those reported by Hailu et al. [5] who found optimal clotting activity of ginger at a pH value of 5.0, a temperature of 65°C, and crude extract concentration of 10% by volume to coagulate camel milk. Grozdanovic et al. [14] reported that kiwifruit extract prepared at pH 5.0 showed patterns of coagulum and whey proteins comparable with those obtained by chymosin using bovine milk. Truc et al. [31] found that optimum temperature for bromelain, pineapple protease, amounts to 10°C-25°C in an acidic environment (pH 4.6-5.2), it refers to 40°C-60°C in a neutral one (pH 6.8-7.1), and only 80% of bromelain remains active at 70°C and pH 3.5-3.8. CaCl 2 concentration has no significant effect on clotting activity. Similar results were mentioned by Castillo et al. [32], who considered that the main effect of CaCl 2 is important on aggregation and firming. us, it is usually added as a texturing agent. Indeed, the presence of ionized calcium is essential for the achievement of the secondary phase of milk coagulation since Ca 2+ ions neutralize negative casein micelle residues to form a firm curd in the second phase of the coagulation process [33].

Proteolytic Activity.
e result of proteolytic activity using different enzymatic extracts compared to chymosin is shown in Figure 2. Figure 2 shows that camel milk with kiwifruit has the lowest proteolytic activity compared to other enzymatic extracts. In the cheese industry, it is still sought that the coagulating enzymes used have high coagulant activity and low proteolytic activity [34]. e proteolytic activity of the extracts depends on several factors, such as the plant source, concentration, and type of protease [35].

Cheese Curd Yield.
Kiwifruit showed the highest curd yield (20.71%, Figure 3) when compared to chymosin and other plant coagulants in camel milk. is result is similar to that of Mazorra-Manzano et al. [21], which mentioned a higher yield using kiwifruit than that of melon and ginger but lowest than chymosin in cow milk. is can be explained by the fact that cheese yield also depends on other factors such as milk quality and composition, heat treatment of milk, type of cheese, and the processing methods used [21].

Plant coagulants have long been considered as possible
substitutes for chymosin in the cheese-making process, but their potential for such use depends on their catalytic properties, stability, and specificity, as these factors can affect cheese yield and sensory properties [36]. In the cheese industry, the choice of the coagulating enzyme is a very determining factor. e most appropriate enzyme is the one with the highest clotting activity. e clotting activity is very variable because it is strongly influenced by the state of maturity of the plant and by the conditions of collection and storage [37].

Physicochemical Characteristics of Curd Cheese.
e physicochemical characteristics of camel cheese with different enzymatic extracts (pineapple, kiwifruit, and ginger) are shown in Table 2.
A significant difference on ash and protein content depending on the type of extract was revealed. e curd obtained with ginger extract showed the lowest (P < 0.05) water content overall although this is still in the range of 60-70% reported for fresh cheese [38,39]. Proteins content was higher in camel milk cheese with pineapple than kiwi and ginger.   e fat-soluble vitamins analyzed were K and E, and the water-soluble vitamins were B5, B7, and B12. e result is shown in Table 3.
Camel cheese with kiwifruit showed higher levels of water-soluble vitamins B7 (375,034 mg/kg), B12, and B5 compared to camel cheese with other enzymatic extracts (pineapple and ginger). e main fat-soluble vitamins detected in camel cheese was vitamin E.
As shown in Table 4, the most predominant mineral element in camel cheese with different enzymatic extracts was Na, K, Ca, Mg, Zn, and Fe. Mineral content of camel cheese with kiwifruit was higher than other extracts especially in Na (605.2 ppm) and Ca (63.11 ppm) content.

Texture Profile Analysis (TPA) of Cheese Curds.
e results of TPA analysis of fresh cheese curds produced from the different plant coagulants are shown in Table 5.
Cheese curd texture varied greatly as a function of the different treatments, ranging from 1.15 to 4.33 MJ (P < 0.05). e highest values (P > 0.05) were obtained for curds made with chymosin and kiwi (4.21 MJ and 4.33 MJ, respectively), while curd made with ginger showed the   Journal of Chemistry lowest value (1.15 MJ). ese results on curd deformation agree with the proteolytic activities of the extracts, which were higher for the ginger extract and lower for the control (chymosin) and kiwi extract (Figure 2). is result was similar to that published by Mazorra-Manzano et al. [21] who found highest curd hardness in kiwi fruits than melon and ginger in cow cheese.

Sensory Evaluation of Curd Cheese.
e sensory quality of cheeses varies according to the manufacturing technology and the chemical and microbiological characteristics of the raw material used. e result of sensory evaluation is shown in Table 6.
Adding enzymatic extract had significant effect on taste, texture, smell, acidity, and color of camel cheese. However, camel cheese with ginger was more acid than other extracts and that with kiwifruit has the best texture. e cheeses obtained are characterized by a slightly bitter taste whatever the extract used. Benani [40] stated that the coagulant agent is characterized by a high proteolytic activity which confers a bitter taste on cheese.
According to the preference test (Figure 4), camel cheese with kiwifruit was preferred by consumer (50%) compared to cheese with the other enzymatic extracts. is result was in agreement with the physicochemical and rheological characteristic of camel cheese made with kiwifruit as coagulant.    Values with different superscript letters within the same column are different (P < 0.05). Values are the mean ± standard deviation.

Conclusion
e kiwi enzymatic extract showed the highest potential for use as a milk-clotting agent in camel milk cheese making since it produced a curd with similar characteristics to those obtained when using commercial chymosin. e textural properties of fresh curds obtained with kiwi and pineapple extracts were similar to those produced using chymosin. e differences observed between plant coagulants and chymosin may have some effect on the texture and flavor of cheeses, opening the possibility for the production of new cheese varieties with additional plant flavors.
Data Availability e datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Imen Fguiri designed and performed the experiments with help of Marzougui Chayma. Imen Fguiri, Moufida Atigui, and Amel Sboui analyzed the data and wrote the manuscript. Samira Arroum and Moahmed Dbara contributed in the experimental analysis. Touhami Khorchani and Mohamed Hammadi revised the paper. : Preference test of camel cheese with different extracts.
CMC P: camel milk cheese with pineapple; CMC K: camel milk cheese with kiwi; CMC G: camel milk cheese with ginger; CMC Chy: camel milk cheese with chymosin.